Method of lyophilizing liposomes

ABSTRACT

Lyophilized liposomal formulations with two or more encapsulated drugs are disclosed. These formulations display superior drug retention profiles and also maintain size distribution following lyophilization and reconstitution.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/891,170 having a filing date of 7 Feb. 2018, which is a continuationof U.S. application Ser. No. 14/352,662 having an international filingdate of 15 Oct. 2012 and which issued as U.S. Pat. No. 10,028,912, whichis the national phase of PCT application PCT/US2012/060293 having aninternational filing date of 15 Oct. 2012, which claims benefit of U.S.provisional patent application No. 61/550,047 filed 21 Oct. 2011. Thecontents of the above patent applications are incorporated by referenceherein in their entirety.

TECHNICAL FIELD

The invention relates to compositions and methods for producinglyophilized liposomes that contain at least two therapeutic ordiagnostic agents that can be stored for prolonged periods of time. Inone aspect, the invention concerns low-cholesterol liposomes optionallyin an external medium comprising a cryoprotectant having resistance tofreeze/thaw and dehydration damage of the liposomes thus preservingtheir size and integrity.

BACKGROUND OF THE INVENTION

Liposomes are closed vesicles having at least one lipid bilayersurrounding an aqueous core. The intra-liposomal space and lipidlayer(s) can entrap a wide variety of substances including drugs,cosmetics, diagnostic reagents, genetic material and bioactivecompounds. Since non-toxic lipids act as the basis for liposomes, theygenerally exhibit low toxicity. The low toxicity coupled with theability of liposomes to increase the plasma circulation lifetime ofagents gives rise to liposomes as vehicles particularly useful fordelivering pharmaceutically active agents. In many cases,liposome-delivered drugs result in superior clinical efficacy pairedwith reduced toxicity.

The practical application of liposomal preparations as drug deliveryvehicles is limited by the chemical and physical stability of thepreparation. Commercialization requires long term stability at both thechemical and physical levels. The use of frozen or freeze-dried(lyophilized) preparations to avoid breakdown of labile drug and/orlipid components provides some improvement in stability. However, duringthe lyophilization process, ice crystal formation can lead to mechanicalrupture, liposome aggregation and fusion (resulting in increasedliposome size). Moreover, when liposomes containing drug are lyophilizedand then reconstituted at room temperature, changes often occur in thestructure of their bilayer(s) which gives rise to accelerated drugleakage.

Prior attempts at preparing lyophilized liposomal compositions haverelied on conventional liposomes which are typically in a liquid phaseat body temperature where movement of the lipids is fluid anduncontrolled. Such conventional liposomes fall into two categories. Thefirst are maintained in a liquid state because they comprise lipidswherein the gel-to-liquid crystalline temperature (T_(c)) is below bodytemperature (i.e., they will be in the liquid phase at bodytemperature). These liposomes are routinely used in the art however; thedownside of being fluid is poor drug retention for many encapsulatedagents.

The second type of conventional liposomes never undergo a liquid to geltransition because they include high amounts of membrane rigidificationagents, such as cholesterol (e.g., 30-45 mol %). Cholesterol acts toincrease bilayer thickness and fluidity while decreasing membranepermeability, protein interactions, and lipoprotein destabilization ofthe liposome. These high amounts of cholesterol are most frequently usedin liposomal studies and historically have been taught as necessary foradequate serum stability and drug retention in vivo, though not alldrugs can be sufficiently retained. Certain drugs exhibit better drugretention both in vitro and in vivo in liposomes containingsubstantially no cholesterol. See, e.g., Dos Santos, et al., Biochim.Biophs. Acta, (2002) 1561:188-201.

On the other hand, liposomes in the gel-phase are more stable andexhibit improved drug retention. The invention takes advantage ofliposomes which are in the gel phase at body temperature (i.e., bodytemperature is below the T_(c) of the liposomes). Gel-phase liposomescan be prepared with a number of lipids; however, those made with moresaturated acyl side chain phosphatidyl lipids, such hydrogenated soy PC,dipalmitoyl phosphatidylcholine (DPPC) or distearoyl phosphatidylcholine(DSPC) are required to have less than 30% cholesterol in order toachieve gel-phases at body temperature. One example of conventionalliposomes that do not exhibit gel-phases at body temperature are thosemade of egg phosphatidylcholine (EPC) which are significantly leaky.

Prior attempts at preparing lyophilized liposomal compositions usingconventional liposomes have involved either empty liposomes or liposomescontaining only a single agent. They may employ a cryoprotectant,typically a saccharide, both inside and outside of the liposomes or alarge osmotic gradient across the liposomal membrane.

For example, cryoprotectants were used to protect against freeze/thawdamage to ‘liquid’ EPC liposomes encapsulating a single agent whenpresent in sufficient amounts both on the inside and outside of theliposomes, ideally when these amounts are equal. See, e.g., U.S. Pat.Nos. 5,077,056 and 4,883,665. The presence of 1%-10% cryoprotectant inthe internal liposomal medium preserves a lyophilized EPCliposome-encapsulated doxorubicin formulation where preferably theinternal osmolarity is near physiological osmolarity. See, e.g., U.S.Pat. No. 4,927,571. Failure to include a cryoprotectant in the liposomeinterior has been shown to result in a loss of liposome integrity uponreconstitution, particularly with regard to retaining an encapsulatedagent. As described, “prevention of leakage requires the sugar bepresent both inside and outside the liposome” (Lowery, M. (June 2002)Drug Development and Delivery, Vol. 2, No. 4).

In one case, protection from vesicle aggregation and fusion, as well asagainst loss of an entrapped drug, has also been reported forhydrogenated soy PC:cholesterol:DSPE-mPEG (51:44:5 molar ratio)liposomes where the liposome preparation contains 44 mol % cholesterolas well as a cryoprotectant and a high concentration of salt in theexternal medium. The presence of 44% cholesterol means that theliposomes will be in the liquid phase at or below body temperature.Furthermore, the protective effect is only realized if a large osmoticgradient exists across the membrane such that the outer liposomeosmolarity is significantly higher than the internal osmolarity. See,e.g., WO01/05372.

Membrane-bound cryoprotectants also further improve resistance tofreezing and lyophilizing of these non-gel phase liposomes. Inparticular, sugars grafted onto EPC or EPC:cholesterol (1:1 molar ratio)liposomal membrane surfaces via oligo(ethylene oxide) linkers consistingof one to three repeating units have been reported to be cryoprotectivefor liposomes containing a fluorescent probe. See, e.g., Bendas, et al.,Eur. J. Pharm. Sci. (1996) 4:211-222; Goodrich, et al., Biochem. (1991)30:5313-5318; U.S. Pat. No. 4,915,951. Baldeschwieler, et al., reportedthat in the absence of the terminal sugar group, liposomes prepared withthe oligoethylene oxide linker itself were unable to protect againstfusion subsequent to freezing. U.S. Pat. No. 4,915,951.

Trehalose in the external medium of a PC liposome formulationencapsulating a single agent provides resistance to liposome aggregationand fusion. U.S. Pat. No. 6,319,517. Other methods of producing smallliposomes stabilized against aggregation require the formation of emptyPC:Cholesterol (1:1 molar ratio) liposomes to which a solution of sugarand a single reagent are added and then subsequently dried. During thedrying process a percentage of the reagent is entrapped within theliposome. These liposomes are reportedly more stable upon storage thanin the absence of sugar. See, e.g., WO99/65465.

As stated previously, most previous techniques for lyophilizationfocused on lyophilization of either empty liposomes or liposomesencapsulating a single agent. Lyophilization with retention of integrityis more challenging where two or more agents are encapsulated,especially if the agents differ in solubility characteristics.Encapsulating two or more agents is often useful since manylife-threatening diseases such as cancer, are influenced by multiplemolecular mechanisms and due to this complexity, achieving cures with asingle agent has been met with limited success. Therefore, almost allcancer treatments involve combinations of more than one therapeuticagent. This is true of treatment of other conditions as well, includinginfections and chronic diseases.

PCT publication WO03/041681, incorporated herein by reference, reportsthat gel-phase liposomes with transition temperatures of 38° C. orgreater can be prepared using saturated phosphatidyl lipids such as DPPCand DSPC and lower amounts (0-20%) of cholesterol if at least 1 mol % ofphosphoinositol (PI) or phosphatidylglycerol (PG) are included in thecompositions. These liposomes, when containing combinations ofencapsulated irinotecan and floxuridine (FUDR) were shown to be stableto freezing at −20° C. Simple freezing is generally less harsh and lessdestructive to liposome integrity than lyophilization.

The use of liposomes as delivery vehicles for these combinations isadvantageous, particularly if the liposomes include, and are capable ofmaintaining, ratios of the agents that are non-antagonistic. Thisgeneral approach is described in detail in U.S. Pat. No. 7,850,990,incorporated herein by reference. This patent teaches how to determinenon-antagonistic or synergistic ratios of various therapeutic agents,including antineoplastic agents that maintain such non-antagonism orsynergy over a wide range of concentrations. The patent also teachesthat it is essential to deliver the drugs in the administered ratio andmaintaining that ratio by letting delivery vehicles control thepharmacokinetics. Exemplified in this patent are liposomes that contain,and maintain the ratio of, non-antagonistic or synergistic ratios of twoor more therapeutic agents, including irinotecan and FUDR. Suchcombinations encapsulated in liposomes would benefit from the advantagesof being stored in lyophilized form if, upon reconstitution, theintegrity of the liposomes and the concentration of the agents and theirratios are maintained. A particularly useful such combination ofcytarabine and daunorubicin encapsulated in liposomes is described inU.S. Pat. No. 8,022,279, also incorporated herein by reference.

The use of these combinations in therapeutic protocols with surprisinglygood results is described in PCT publication WO2007/050784 and PCTpublication WO2008/101214. Additional formulations with liposomalencapsulation of desired drug delivery options are described inWO2009/097011 and WO2009/070761, as well as WO2010/043050. Theseformulations are simply exemplary of useful compositions wherein two ormore therapeutic agents are contained in liposomes for delivery to thepatient.

As described above, preparing stable lyophilized compositions ofliposomes in general that maintain their integrity upon reconstitutionhas been difficult and unpredictable. Obtaining such stable liposomalcompositions for combinations of two or more agents is even morechallenging. Thus, the success of the method of the invention inobtaining lyophilized liposomes wherein the liposomes contain two ormore therapeutic or diagnostic agents, and wherein they maintain theirintegrity upon reconstitution, is a remarkable achievement.

DISCLOSURE OF THE INVENTION

It has consistently been reported that a cryoprotectant is required bothinside and outside of liposomes in order to maintain liposome integrityupon reconstitution after lyophilization, particularly in order toensure retention of an encapsulated agent. The present inventors haveidentified stable liposomes that require no internal cryoprotectant forsuccessful lyophilization of liposomes encapsulating not only one, buttwo or more active agents.

The invention relates to successful lyophilized gel-phase liposomalpreparations that contain more than one therapeutic and/or diagnosticagent and no internal cryoprotectant. Thus, in one aspect, the inventionis directed to a lyophilized liposomal composition wherein saidliposomes are stably associated with at least two therapeutic and/ordiagnostic agents and wherein when said composition is reconstituted,the mean diameter of the liposomes is maintained compared to thepre-lyophilization state and the percentage of each of the agents thatremains encapsulated in the liposomes is maintained at a satisfactorylevel. The integrity of the liposomes is thus measured as the percentageof encapsulated agents retained after reconstitution of the liposomes.An additional parameter used as a criterion for satisfactorylyophilization is minimal change in size distribution. A particularlyimportant embodiment is that wherein the agents are encapsulated insidethe liposomes at a defined ratio and wherein the ratio of these agentsis maintained when the lyophilized forms are reconstituted.

Typical conditions for achieving this result include the use ofgel-phase liposomes with gel-to-liquid crystalline transitiontemperatures (T_(c)) that are at least room temperature and may be at orabove human body temperature. Body temperature is considered to be about37° C. The liposomes may be low cholesterol liposomes that arestabilized with phosphatidylglycerol and/or phosphoinositol. Theliposomes contain substantially no internal cryoprotectant, but maycontain external cryoprotectant at their surfaces and thus may belyophilized in the presence of a medium containing cryoprotectant. Theterm “substantially no internal cryoprotectant” is meant to includeliposomes that comprise no internal cryoprotectant as well as liposomeswhich contain an amount of cryoprotectant which does not affect thefreezing and/or lyophilization process of said liposomes (i.e., 125 mMcryoprotectant or less that is, an “inactive” amount). Therefore“substantially no internal cryoprotectant” is defined to be from about0-125 mM cryoprotectant inside the liposomes. It is important to notethat preventing drug leakage following the lyophilization process issignificantly more difficult than retention of liposome size. Asmentioned above, drug retention following lyophilization hashistorically been achieved via the use of a cryoprotectant both on theinside and outside of the liposomes.

Thus, in one embodiment, the liposomes have gel-to-liquid crystallinetransition temperatures (T_(c)'s) of the membrane greater than roomtemperature or greater than 25° C. or 37° C. so that, at least at roomtemperature, e.g., 25° C., the lipid membrane is sufficiently gel-liketo assist in retaining the drugs. The compositions afford retention ofencapsulated agents, and reduced aggregation and fusion uponlyophilization and reconstitution, thereby providing useablecompositions with extended shelf life. The enhanced protection from thelyophilization process is independent of osmotic potential. Theseliposomes maintain their size distribution and drug-encapsulationprofiles over extended periods of time under pharmaceutically relevantconditions.

Methods to prepare the lyophilized liposome compositions thus mayinclude a cryoprotectant external to the liposomes at a selectedconcentration wherein the liposome membrane prior to freezing andlyophilization is below its phase transition temperature T_(c).Preferably, the liposomes are frozen at a temperature which is below theglass transition temperature (T_(g)) of the solution which comprises theliposomes with encapsulated drug as well as the extraliposomal mediumwhich contains the cryoprotectant.

The invention is also directed, in other aspects, to methods ofpreparing lyophilized liposomes containing two or more therapeuticand/or diagnostic agents according to the embodiments set forth above,to methods of reconstituting said lyophilized compositions, and tomethods of administering the reconstituted liposomes to animals, and tomethods of treating animals affected by, susceptible to, or suspected ofbeing affected by a disorder (e.g., cancer).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a particle size profile of CPX-1 liposomes before freezing.

FIG. 2 shows a particle size profile of reconstituted CPX-1 liposomesimmediately after freezing, lyophilizing and reconstitution.

FIG. 3 shows a particle size profile of reconstituted CPX-1 liposomes 1month after storage.

FIGS. 4A-4C show particle size profiles of reconstituted CPX-1 liposomes6 months after storage.

MODES FOR CARRYING OUT THE INVENTION

The invention provides, for the first time, lyophilized gel-phaseliposomal compositions that contain two or more therapeutic and/ordiagnostic agents such that the characteristics and properties of thereconstituted lyophilized composition essentially match those of thecomposition prior to lyophilization. These characteristics may includethe mean diameter, size distribution, and contents of the liposomes. Thecontent of the liposomes refers to the retention of the agents; in someembodiments, the ratio of the agents is retained as well.

Although the liposomes contain therapeutic and/or diagnostic agents, inthe present application, “drugs” is sometimes used as a shorthand todesignate these.

The gel-phase liposomes comprise one or more lipid bilayers enclosing aninternal compartment. These liposomes can be bi-lamellar or unilamellarvesicles. Unilamellar liposomes (also known as unilamellar vesicles or“ULV”) enclose a single internal aqueous compartment and are classifiedas either small unilamellar vesicles (SUV) or large unilamellar vesicles(LUV). LUV and SUV range in size from about 50 to 500 nm and 20 to 50nm, respectively. Bilamellar liposomes have two lipid membranes whereinthe inner membrane surrounds a single internal aqueous compartment andthe second, larger outer membrane surrounds the inner membrane thuscreating a second internal aqueous compartment.

Maintaining the size distribution of the gel-phase liposomes may beassessed experimentally by obtaining particle size profiles such asthose set forth in FIGS. 1-4 herein. Size distribution determined byquasielastic light scattering is typically presented as a histogramshowing the mean diameter of the liposomes. Significant sizedistribution measurements most commonly used in the art are D10, D90,D99 or a standard deviation or polydispersity index. “D99” valuessignify that 99% of the liposomes are less than a referenced size ormore than a referenced size. This is particularly useful if, forexample, it is important to exclude either an upper or lower size. Forexample, in certain embodiments it is desirable to ensure that noliposomes over 200 nm in mean diameter are present.

A specific example which has a D99 value of 178 nm is used toillustrate. A D99 value measuring 178 nm (as seen in Table 1 of Example2) ensures that at least 99% of the liposome population is less than 178nm. The D10 and D90 values for mean diameters, also commonly used, arethose in which no more than 10% of the population is smaller than aminimum referenced size (i.e. D10) and for D90, where 90% of thepopulation is at or less than an upper referenced size limit. Forexample, as seen in Batches 1 and 2, the D10 value is 68 nm such that nomore than 10% of the liposome population is less than 68 nm. The D 90value shows that 90% of the population is at or less than 135 or 137 nmfor Batches 1 and 2, respectively. Maintaining the size distribution ofthe liposomes after lyophilization and reconstitution is defined hereinas demonstrated by showing that the referent value of a selected D valuechanges by no more than 50%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6% or 5%upon reconstitution compared to its value before freezing and/orlyophilization. The D values selected may be 99, 98, 94 and interveningintegers to 90 or D10.

One characteristic of the lyophilized liposomes relates to the meandiameter of the liposomes in the composition. The mean diameter of aliposomal composition is maintained on reconstitution when the meandiameter of the liposomes does not increase more than 50%, 25%, on avolume weighted basis 20%, 15%, 10%, 9%, 8%, 7%, 6% or 5% afterlyophilization and upon reconstitution based on the diameter beforefreezing. A concomitant value, such as a 10% increase in mean liposomaldiameter coupled with a 10% increase in the referent for D90 (or other Dvalue such as those listed above) is one measure to assure that theparticle (e.g., liposomal) size distribution has not changed. Theoverall nature of the distribution can also be assessed preferably on avolume weighted basis, as shown in FIGS. 1-4.

In more detail, a composition of liposomes contains a range of sizestypically following a Gaussian curve. The mean diameter of the liposomesmay increase by no more than about 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%,6% or 5% from its original size upon reconstitution after freezing orlyophilization and reconstitution. For example, a sample of liposomeswhose mean diameter is 90 nm would be considered to resist the effectsof freezing and/or lyophilization if, upon reconstitution, the meandiameter is no more than 30% greater, i.e., about 117 nm. Size increasesgreater than these suggest that aggregation and fusion of the liposomeshas occurred. A sufficiently sensitive measuring technique may beemployed for measuring changes in size distribution or mean diameter sothat changes of less than 10% can be measured.

Another criterion for preservation of integrity is retention of theencapsulated agents. Unlike mean diameter, size distribution and drugratio, which are evaluated relative to pre-lyophilization values, drugretention is evaluated relative to total drug per se afterreconstitution, i.e., based on the total drug in the lyophilizedcomposition. The percentage of drug encapsulated inside the liposomes orthe percentage of drug in the external medium outside of the liposomes(% “unencapsulated”) are relative to the total amount of drug in thecomposition. In one embodiment, at least about 75% of the encapsulatedagents is retained as encapsulated after lyophilization and uponreconstitution. At least about 85% of each may be retained asencapsulated and or at least about 90%, or 95%. This can similarly bemeasured by the amount of unencapsulated drug in the surrounding mediawhich should not be more than 25%, 20%, 15%, 10% or 5% of the originalamounts encapsulated upon reconstitution of the lyophilized liposomes.

The ratio of encapsulated therapeutic and/or diagnostic agents ismaintained on reconstitution if the ratio does not vary by more than20%, 10%, 9%, 8%, 7%, 6%, or 5% from the ratio in the pre-lyophilizedcomposition itself. Ratios are expressed as molar ratios.

In one embodiment, the mean diameter of the liposomes afterlyophilization and upon reconstitution of said liposomes will increaseby no more than 25% as compared to said value measured prior tolyophilization. In another embodiment, the mean diameter of theliposomes after lyophilization and upon reconstitution of said liposomeschanges by no more than 15% as compared to said value measured prior tolyophilization. In still other embodiments, the mean diameter of theliposomes after lyophilization and upon reconstitution of said liposomeschanges by no more than 10%, 9%, 8%, 7%, 6%, or 5% as compared to saidvalue measured prior to lyophilization.

In some embodiments, the percent of unencapsulated drug is no more than25% of that originally encapsulated upon reconstitution of saidliposomes. In other embodiments, the percent of unencapsulated drug isno more than 15% of that originally encapsulated upon reconstitution ofsaid liposomes. In other embodiments, the percent of unencapsulated drugis no more than 10%, or is no more than 9%, 8%, 7%, 6% or 5% of thatoriginally encapsulated upon reconstitution of said liposomes.

Stated another way, in some embodiments, the percent of the encapsulateddrugs retained is no less than 75% upon reconstitution of saidliposomes. In other embodiments, the percent of each encapsulated drugis no less than 85% or 90% or 95% upon reconstitution of said liposomes.

Combinations of these parameters are also included. For example, themean diameter may increase no more than 25%, and the percentage ofencapsulated drug be at least 90%, or the mean diameter may increase nomore than 10% and the percentage of encapsulated drug at least 90%.

In some embodiments, the size distribution of the liposomes changes byno more than 25% after lyophilization and upon reconstitution of saidliposomes as compared to prior to lyophilization. In other embodiments,the size distribution of the liposomes changes by no more than 15%, 10%,9%, 8%, 7%, 6%, or 5% after lyophilization and upon reconstitution ofsaid liposomes as compared to prior to lyophilization.

As noted above, various combinations of these parameters or criteria forsuccessfully lyophilizing and reconstituting liposomes arecontemplated—e.g., at least 85% encapsulated drugs combined with no morethan 15% increase in mean diameter optionally combined with no more than5% change in size distribution. Each of the possible combinations ofthese parameters is within the scope of the invention.

Gel-phase liposomes can be generated by conventional techniques, e.g.,the ether injection method (Deamer, et al., Acad. Sci. (1978) 308:250),the surfactant method (Brunner, et al., Biochim. Biophys. Acta (1976)455:322), the freeze-thaw method (Pick, et al., Arch. Biochim. Biophys.(1981) 212:186) the reverse-phase evaporation method (Szoka, et al.,Biochim. Biophys. Acta. (1980) 601:559-571), the ultrasonic treatmentmethod (Huang, et al., Biochemistry (1969) 8:344), the ethanol injectionmethod (Kremer, et al., Biochemistry (1977) 16:3932), the extrusionmethod (Hope, et al., Biochim. Biophys. Acta (1985) 812:55-65) and theFrench press method (Barenholz, et al., FEBS Lett. (1979) 99:210).

These processes can be used in combination. Small unilamellar vesicles(SUVs) in particular can be prepared by the ultrasonic treatment method,the ethanol injection method and the French press method. Largeunilamellar vesicles (LUVs) may be prepared by the ether injectionmethod, the surfactant method, the freeze-thaw method, the reverse-phaseevaporation method, the French press method or the extrusion method.Preferably, LUVs are prepared according to the extrusion method.

The lyophilization and reconstitution are conducted under conditionswherein the liposomes are in the gel phase. The gel-to-liquid transitiontemperature of the liposomes should therefore be greater than roomtemperature, i.e., about 20-30° C. and more preferably, at or above bodytemperature. Room temperatures may vary considerably, but it isimportant that the lyophilization process begin under conditions wherethe liposomes are in a gel state. In some embodiments, the T_(c) is atleast as high as body temperature (i.e., about 37° C.). In someembodiments, the liposomes are prepared at a temperature below the phasetransition temperature in order to maintain the gel-like state. Anysuitable internal medium may be employed. Typically, the internal mediumis an aqueous medium. The internal medium contains substantially nocryoprotectant (i.e., less than 125 mM cryoprotectant). The internalmedium may contain less than 100 mM cryoprotectant, or less than 50 mMcryoprotectant, or no cryoprotectant.

Liposome formulations which have suitable T_(c) values may be “lowcholesterol” liposomes, i.e., those prepared in the presence of, andcontaining an amount of cholesterol that is insufficient tosignificantly alter the phase transition characteristics of theliposome, i.e., typically 20 mol % or less cholesterol. Greater than 20mol % of cholesterol broadens the range of temperatures at which phasetransition occurs, with phase transition disappearing at highercholesterol levels. A liposome having low cholesterol will have lessthan 20 mol % or less than 15 mol % cholesterol, or 10 mol % or 5 mol %or less cholesterol or be free of cholesterol. Such liposomes optimallyrequire at least 1 mol % of a stabilizing agent such as PG or PI.

In those methods where cryoprotectant is used, the cryoprotectantpreferably is present only in the external medium of the formulation.Typically, the cryoprotectants are disaccharides such as sucrose,maltose, trehalose and lactose. The cryoprotectant may be a disaccharidesuch as sucrose having a concentration that is about 100 mM to 500 mM orabout 250-400 mM, or above 300 mM. The external medium may contain about100 mM to 500 mM cryoprotectant and the internal medium contain lessthan 125 mM cryoprotectant or the external medium contains about 250 mMto 400 mM cryoprotectant and the internal medium contains less than 100mM cryoprotectant or the external medium contains about 250 mM to 400 mMcryoprotectant and the internal medium contains less than 50 mMcryoprotectant or the external medium contains about 250 mM to 400 mMcryoprotectant and the internal medium contains no cryoprotectant. Thecryoprotectant may be a saccharide, such as sucrose.

The gel-phase liposomal formulations can be lyophilized or freeze-driedusing any appropriate protocol. The initial temperature of thelyophilization chamber is preferably below the glass transitiontemperature (T_(g)) of the solution which comprises the external mediumas well as containing the liposomes with encapsulated drugs. Forexample, the liposomes may be frozen at a temperature below about −5°C., or below about −10° C., or below about −20° C., or below about −30°C., or below about −40° C. In some embodiments, when sucrose is used asthe cryoprotectant solution, the initial temperature of thelyophilization chamber is less than −32° C. which is the T_(g) ofsolution of sucrose. “T_(g)” includes the “glass transition temperature”and “glass phase transition temperature” which is the approximatemidpoint temperature at which the unfrozen solution undergoes atransition from a soft, viscous gel to a hard and relatively brittleform.

The lyophilized liposomes may be stored at or below room temperature.Some exemplified embodiments have liposomes which are stored at or below5° C. Some other exemplified embodiments have liposomes which are storedat 25° C. The lyophilized product remains stable (e.g., retains itsrelative particle size and maintains encapsulated drug) for at leastabout six months, or at least about nine months, or at least about oneyear, or at least about twenty-four to thirty-six months.

The entrapped agents are therapeutic or diagnostic agents, oftenanticancer agents. Remarkably, the contents and integrity of thegel-phase liposomal compositions are maintained even though the agentsdiffer in their solubility characteristics with respect to aqueous andnonaqueous solvents. Using the approach of the invention, agents thatdiffer in log partition coefficient (Log P) by as much as 1.0 may besuccessfully retained. Differences in log partition coefficient of 1.5or 2.0 or 3.0 may be tolerated as well. One of the agents may beamphipathic while the other is water-soluble or one may be hydrophobicwhile the other is water-soluble. The Log P values are based on thepartition coefficients between octanol and water—i.e., are the logarithmbase 10 of the ratio of amount in octanol to the amount in water whenthe compound is subjected to phase separation.

The anticancer agents may include an anthracycline (for example,daunorubicin, doxorubicin, epirubicin or idarubicin). These agents areamphipathic. The anticancer agents may include a nucleoside analog forexample, cytosine arabinoside, 5-FU or FUDR which are hydrophilic. Otheranticancer agents include camptothecin or camptothecin derivative, suchas irinotecan which are amphipathic. Both an anthracycline and anucleoside analog are encapsulated in some cases or both a camptothecinor camptothecin derivative and a nucleoside analog are encapsulated.Encapsulation and/or loading of agents into liposomes may be carried outusing any suitable loading techniques including passive and activeloading. Important embodiments include those described in the abovecited U.S. Pat. Nos. 7,850,990 and 8,022,279—i.e., combinations ofirinotecan:floxuridine (FUDR) at 1:1 molar ratio anddaunorubicin:cytarabine (AraC) at 1:5 molar ratio. Particularformulations of these drugs are designated CPX-1 and CPX-351,respectively.

The drugs are incorporated into the aqueous internal compartment(s) ofthe liposomes either by passive or active loading procedures or somecombination thereof. In passive loading, the biologically active agentcan be simply included in the preparation from which the liposomes areformed or, alternatively, the active agent can be added to the outsideof preformed liposomes and loads passively down its concentrationgradient into the liposomes. Optionally, unencapsulated material may beremoved from the preparation by any suitable procedures. Alternatively,active loading procedures can be employed, such as ion gradients,ionophores, pH gradients and metal-based loading procedures based onmetal complexation. One embodiment commonly employed for suitable drugsis loading via metal-based procedures.

The liposomes are about 80-500 nm in size. In one embodiment, theliposomes have a diameter of less than 300 nm, sometimes less than 200nm. In one example, the nominal size of these liposomes is approximately100 nm. In some embodiments, the liposome membrane is composed ofdistearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol(DSPG) and cholesterol (CHOL). In some embodiments, the liposomemembrane is composed of 50-80% DSPC, 1-20% DSPG and 1-20% CHOL. In otherembodiments, the liposome membrane is composed of 50-80% DSPC or DPPC,1-20% DSPG or distearoylphospatidylinositol (DSPI), 1-20% CHOL and theliposomes contain less than 125 mM cryoprotectant in the intraliposomalmedium. In some exemplified embodiments, the liposome membrane iscomposed of 50-80% DSPC or DPPC, 1-20% DSPG or DSPI, 1-20% CHOL and theliposomes contain less than 50 mM cryoprotectant in the intraliposomalmedium. In other exemplified embodiments, the liposome membrane iscomposed of DSPC, DSPG and CHOL in about a 7:2:1 molar ratio and containno cryoprotectant in the internal liposomal medium. In one instance, theliposomes are prepared by a water-in-oil derived liposome method andextruded liposomes are suspended in phosphate-buffered sucrose at pH7.0. In another instance, the extruded liposomes are suspended insucrose. In one embodiment, the extruded liposomes are suspended in250-400 mM sucrose.

Any suitable means of encapsulating the drug combination in theliposomes can be employed. In a specific embodiment, irinotecan andfloxuridine are co-loaded into DSPC/DSPG/CHOL (7/2/1) preformedliposomes whereby floxuridine is passively loaded into the liposomes andirinotecan is actively loaded at 50° C. using copper sulphate or coppergluconate in the internal medium. See co-owned U.S. Pat. Nos. 7,850,990and 7,238,367 both of which are incorporated by reference. In anotherspecific embodiment, cytarabine and daunorubicin are encapsulated in theliposome whereby the cytarabine is passively encapsulated into preformedliposomes and the daunorubicin is actively accumulated inside theliposomes at high trapping efficiencies using a coppergluconate/triethanolamine-based loading procedure. See, e.g., copendingand co-owned PCT Applications WO05/102359 and WO07/076117A2 both ofwhich are also incorporated by reference in their entirety.

The lyophilized compositions of the invention provide convenience instorage, preservation, and ease of shipping. These lyophilizedcompositions retain their characteristics over long periods of time.

For use, the compositions of the invention are reconstituted in asuitable pharmaceutical carrier or medium.

These formulations for use are prepared according to standardreconstitution techniques using a pharmaceutically acceptable carrier.Generally, normal saline will be employed as the pharmaceuticallyacceptable carrier. Other suitable carriers include, e.g., water,buffered water, dextrose, 0.4% sodium chloride, 0.3% glycine, and thelike, including glycoproteins for enhanced stability, such as albumin,lipoprotein, globulin, etc. These compositions may be sterilized byconventional, well known sterilization techniques. The resulting aqueoussolutions may be packaged for use or filtered under aseptic conditionsand lyophilized, the lyophilized preparation being combined with asterile aqueous solution prior to administration. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, etc. Additionally, the liposome suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as alphatocopherol and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

The reconstituted formulations may be administered to animals, includinghumans, or other mammalian species, such as non-human primates, dogs,cats, cattle, horses, sheep, and the like, and may be used to treat avariety of diseases. Examples of medical uses of the compositions of thepresent invention include but are not limited to treating cancer,treating cardiovascular diseases such as hypertension, cardiacarrhythmia and restenosis, treating bacterial, fungal or parasiticinfections, treating and/or preventing diseases through the use of thecompositions of the present inventions as vaccines, treatinginflammation or treating autoimmune diseases. For treatment of humanailments, a qualified physician will determine how the compositions ofthe present invention should be utilized with respect to dose, scheduleand route of administration using established protocols. Suchapplications may also utilize dose escalation should bioactive agentsencapsulated in liposomes and lipid carriers of the present inventionexhibit reduced toxicity to healthy tissues of the subject.

The pharmaceutical compositions are typically administered parenterally,e.g., intravenously, but other routes may be employed. Dosage for theliposome formulations will depend on the ratio of drug to lipid and theadministrating physician's opinion based on age, weight, and conditionof the patient.

Overall, one process useful in the invention comprises lyophilizing acomposition of liposomes wherein said liposomes comprise 20 mol % orless cholesterol and two or more active agents, and wherein the liposomemembrane is below its phase transition temperature when at roomtemperature and in an external medium that contains a cryoprotectant;storing the lyophilized liposomes; and reconstituting the lyophilizedliposomes in a predetermined aqueous volume. The liposomes arelyophilized at a temperature below about −5° C., or below about −10° C.,and below about −20° C., or even below about −30° C. or −40° C., and canbe stored at or below room temperature (about 23-25° C.).

In one embodiment, the liposome composition is comprised of 2-20%cholesterol, or at any intermediate value such as 10% cholesterol.

In one embodiment, the lyophilized composition comprises liposomescomprised of about 10% cholesterol, a disaccharide at a selectedconcentration in the external medium where reconstitution, performed atroom temperature, is below the T_(c), and wherein the cryoprotectant isunbound and present on the outside only of the liposomes.

In another embodiment, the lyophilized liposome composition comprisingtwo or more encapsulated drugs upon reconstitution with a predeterminedvolume of aqueous medium, yields a liposome composition comprising: (a)liposomes containing 20 mol % or less cholesterol, (b) liposome sizespredominantly between about 80-500 nanometers, (c) liposome-entrappedagent(s) wherein the percent encapsulation of said agent(s) is not lessthan about 95%, 90%, 85%, 80% or 75%; and (d) between about 100 mM-500mM cryoprotectant in the external liposomal medium. In some embodiments,between 250-400 mM cryoprotectant is present in the external liposomalmedium. In some embodiments, about 9.5-10% cryoprotectant is present inthe external liposomal medium.

In one embodiment, unilamellar or di-lamellar gel-phase liposomescomprising 20 mol % or less cholesterol, at least two drugs and at leastabout 300 mM sucrose on the outside of the liposomes are lyophilized andupon reconstitution at least about 90% of each of the encapsulated drugsis encapsulated and the mean liposome diameter changes by less thanabout 25%.

As used herein, “a” or “an” means “at least one” or “one or more,”unless it is clear from the context that only a single referent isintended.

The following examples are provided solely to illustrate but not tolimit the invention.

Example 1 Lyophilization of CPX-1

Irinotecan and floxuridine 1:1 are co-encapsulated inDSPC/DSPG/Cholesterol (7:2:1 mol ratio) liposomes and designated CPX-1.Lyophilized CPX-1 resulted in stable drug-loaded liposomes, such that,there was minimal leakage of active pharmaceutical ingredients from thereconstituted dosage form. Irinotecan hydrochloride, used in CPX-1, hasa predicted log partition coefficient (Log P) of 3.94. Floxuridine has apredicted Log P of −1.14.

Thermal analyses were generated for CPX-1 liposomal drug product usingvarious lots to provide information on the glass transition temperature(T_(g)), change in heat capacity, and other exothermic events. Thecollapse temperature of CPX-1 liposomal drug product was determined byfreeze dry microscope for two lots. These results were employed indetermining the final lyophilization cycle.

The samples consisted of a bluish-green bulk aqueous suspensionformulated with liposomes containing a 1:1 ratio of two activepharmaceutical ingredients, irinotecan hydrochloride and floxuridine.Samples were stored at −20° C. (or in some cases −80° C.) with ambientrelative humidity, and thawed overnight in a refrigerator and mixedthoroughly prior to filling and lyophilization.

Cycle 1: Using a 20-mL Class A pipette, 20 cc of CPX-1, was filled into60 cc glass molded vials. Twenty-four vials were loaded into a LyoStarIITray Dryer with two product vials fitted with thermocouple probes torecord product temperature and were freeze dried over four and one-halfdays. After backfilling the vials with nitrogen gas to a chamberpressure of about 720,000 mTorr, the vials were stoppered, removed fromthe Tray Dryer and labeled as Lot TP-CPX1-001-032405. Several of thelyophilized vials were then placed on accelerated stability at 25° C.and 40° C. with the remaining vials being stored at −20° C.

Cycle 2: Approximately 21-mL of CPX-1, were filled into 60 cc glassmolded vials and 50 cc glass tubing vials, respectively. The vials wereloaded into a LyoStarII™ Tray Dryer with one thermocouple probe in aproduct vial on the top shelf and one in a product vial on the bottomshelf. Upon completion of the lyophilization cycle, the vials werebackfilled with nitrogen gas to a chamber pressure of about 720,000mTorr, stoppered, removed from the Tray Dryer, and labeled as LotTP-CPX1-002-041305T. Several of the lyophilized vials were then placedon accelerated stability at 25° C. and 40° C. with the remaining vialsbeing stored at −20° C.

Cycle 3: The samples for lyophilization were prepared in a similarmanner as for Cycle 2 except only filled into 50 cc glass tubing vials.The sealed vials were labeled as CPX-1 Drug Product, LotTP-CPX1-003-051105T. Several of the lyophilized vials were then placedon accelerated stability at 25° C. and 40° C. with the remaining vialsbeing stored at −20° C.

Cycle 4: The samples for lyophilization were prepared in a similarmanner as for Cycle 2 except CPX-1, was filled into 50 cc glass tubingvials. The sealed, lyophilized vials were labeled as CPX-1 Drug Product,Lot TP-CPX1-004-051805T and stored for stability studies at −20° C., 5°C., 25° C., and 40° C.

Cycle 5: The samples for lyophilization were prepared in a similarmanner as for Cycle 2 were filled into 50 cc glass tubing vials. Thesealed, lyophilized vials were labeled as CPX-1 Drug Product TP-CPX1-005062705T-300. The CPX-1 Liposomal Drug Product vials were stored instability chambers at −20° C., 5° C., and 25° C.

First Lyophilization Cycle Run.

The primary goal for the first lyophilization cycle run (Cycle 1) was todetermine if the formulated bulk CPX-1 liposomal drug product (CPX-1)could be successfully freeze dried with a gentle, two-step primary dryphase. The success of this lyophilization run was gauged by analyzingthe drug product's temperature and pressure profiles and by visuallyinspecting the appearance of the lyophilized cakes.

The lyophilization product profile for cycle 1 showed that the bulk icewas removed during the −10° C. primary dry step. This was evident inthat the product temperature slightly exceeded the shelf temperature.Also, the thermocouple pressure, which measures true pressure plus thepartial pressure of water vapor, decreased toward that of thecapacitance manometer pressure, or true pressure. In addition, thelyophilized drug product vials appeared dry with little, or no evidenceof cake collapse. However, some analyte concentration or stratificationwas observed. To optimize the cycle, the thermal treatment phase and theprimary drying steps were altered for the second run.

Second Lyophilization Cycle Run.

The second lyophilization run (cycle 2) was conducted using a similargentle primary and secondary dry phases as cycle 1. To maximize the loadof ice in the lyophilizer, vials filled with deionized water were loadedinto unoccupied shelf space. The success of the lyophilization run wasalso gauged by the temperature and pressure profiles and by visuallyinspection of the lyophilized cakes.

The drug product in the 50 cc tubing vials appeared to freeze dry in amore homogenous manner even though the product temperature and pressureprofiles for the 50 cc glass tubing vial and the 60 cc glass molded vialover the four and one-half day cycle were similar. About eight andone-half hours after reaching the secondary dry shelf temperature, theproduct temperature displays completion of the bulk ice sublimation bycrossing over the ice barrier (i.e., the product temperature exceeds 0°C.). However, these vials were not sufficiently dried. The producttemperature was below the shelf temperature at the end of the primarydry phase, and the difference between the thermocouple pressure and thecapacitance manometer pressure was unchanged from the beginning of therun through the end of the secondary dry phase, which indicates thepresence of substantial bulk ice in the vials.

Because the shelf temperatures employed in the primary dry phase failedto impart enough energy to drive the product sublimation rate towardcompletion, a third lyophilization cycle was developed to drive theprimary drying phase to completion by using a shelf temperature andchamber pressure that increases ice sublimation, while not exceeding thecollapse temperature.

Third Lyophilization Cycle Run.

Based on the results from cycle 2 and the thermal analyses, the shelftemperature and chamber pressure for cycle 3 were adjusted to facilitateprimary drying, while maintaining product temperatures below theestimate collapse temperature of −20° C. To maximize the load of ice,the cycle was conducted under fully loaded run conditions.

The profile plot obtained for Cycle 3 showed that the initial primarydry shelf temperature, −20° C. at 100 mTorr pressure, did not drive thesublimation of bulk ice sufficiently in 40 hours under fully loaded runconditions. Also, the thermocouple pressure trace did not significantlydecrease toward the capacitance manometer pressure until the end of the−10° C. primary dry phase due to the limited duration of this phase.However, the profile demonstrated that the −10° C. second primary dryshelf temperature and duration was able to maintain the producttemperature below the collapse temperature of −20° C. until all of thebulk ice sublimated, which was evident in the rapid increase in producttemperature to eventually exceed the shelf temperature at the end of thephase.

Fourth Lyophilization Cycle Run.

To finalize the shelf temperature(s) for the primary drying phase, thefourth lyophilization cycle (cycle 4) employed a shelf temperature of−10° C. for a longer duration with a 6-hour initial primary dry step at−20° C. under fully loaded run conditions.

Based on the lyophilization cycle profile and visual observation, the−20° C. shelf temperature for the initial primary dry phase appeared tohave little benefit in drying the vials. The product temperature probesexceeded shelf temperature at −10° C. following a hold time of about 60hours. The thermocouple pressure during the secondary drying phaseindicated that the vials had relatively low residual moisture, since theproduct temperature profile closely matched that of the shelftemperature.

The encapsulation of drug substances, liposomal particle size, andaverage residual moisture was evaluated for vials of the lyophilizedproduct. The Karl Fischer method employed recovered an average residualmoisture content of 3.1%, which was not an overly dry liposomal product.Also, the analyses for particle size and percent encapsulation ofirinotecan found that the lyophilized product was unchanged compared tothe pre-lyophilized material. However, the percentage of unencapsulatedfloxuridine increased from 7.0% in the pre-lyophilized bulk to 8.6% inthe lyophilized product when stored at −20° C. for 13 weeks. Also, afterstressing the product for four weeks at 25° C., the percentage ofunencapsulated floxuridine increased to 11.8%, which exceeded thetentative specification of less than 10% unencapsulated floxuridine forthis drug product.

Fifth Lyophilization Cycle Run.

The goal of the fifth lyophilization cycle run (Cycle 5) was to decreasethe shelf temperature for the secondary drying phase from +20° C. to+10° C. in order to minimize Floxuridine leakage while achieving asuitable residual moisture under fully loaded run conditions. Themoisture content for this material was assessed during the secondary dryphase by periodically performing a pressure rise measurement.

The drug product profile for cycle 5 showed that the bulk ice waslargely sublimated following 72 to 84 hours of primary drying at −10° C.Furthermore, the material appears to been dried sufficiently byemploying a shelf temperature of +10° C. at 50 mTorr for 12 hours, basedupon the pressure detector differences and the pressure rise studies.

To evaluate the suitability of this lyophilized material, thereconstitution time for lyophilized drug product vials from both cycles4 and 5 were evaluated using 19 mL of water injected through thestoppers with an 18 gauge needle and a 30 cc syringe. The averagereconstitution time was determined to be 40 and 93 seconds for cycles 4and 5, respectively. Furthermore, the Karl Fischer results for cycle 5recovered an average residual moisture of 3.2%, which was in goodagreement with that of vials from cycle 4.

The encapsulation of the drug substances and liposomal particle sizewere also evaluated. For the cycle 5 lyophilized drug product, thepercentage of unencapsulated irinotecan was 0.4% at −20° C. after 7weeks of storage and 0.9% at 25° C. after 4 weeks of storage. Theparticle size for the lyophilized drug product increased only slightlyafter 8 weeks of storage at 5° C. compared to the drug product whenstored at −20° C., but increased nearly 10 nm after only 4 weeks ofstorage at 25° C. Unsurprisingly, the percentage of unencapsulatedfloxuridine showed a similar trend to the particle size changes. Thepercentage of unencapsulated floxuridine was 5.5% at −20° C. after 7weeks, 7.7% after 8 weeks at 5° C., and 18.7% at 25° C. after 4 weeks.

The fifth lyophilization cycle run, which employed a decreased shelftemperature during its secondary dry phase, succeeded in producingacceptable lyophilized CPX-1 liposomal drug product with increasedretention of encapsulated product.

Example 2 Particle Size Profile Over Time Remains Unchanged inLyophilized Liposomes

Experiments were conducted in order to examine the impact of freezing,lyophilization and storage on the size distribution of dual-loaded CPX-1and CPX-351 liposomes. CPX-351 is a formulation of daunorubicin andcytarabine at a mole ratio of 1:5 in liposomes that are distearoylphosphocholine (DSPC): distearoyl phosphatidylglycerol (DSPG): andcholesterol (CHOL) at a mole ratio of 7:2:1. Daunorubicin has apredicted Log P of 1.68. Cytarabine has a predicted Log P of −2.17.

The particle size distribution of liposomes co-loaded was measuredbefore and after freezing and lyophilization the liposomes as well asafter one and six months of storing the lyophilized preparations.

CPX-1 liposomes were prepared with an external buffer of 300 mM sucrose,20 mM phosphate, pH 7.0. Aliquots of 900 μl were added into 2 mL vialswere placed into a metal pan (pre-cooled to −20° C.) and stored at −20°C. overnight. After freezing, the samples were moved to the lyophilizer(pre-cooled to −20° C.). The vacuum was applied and the shelftemperature was maintained at −20° C. for 7 hours and subsequentlyincreased to −10° C. for approximately 16 hours. For a third temperaturestep, the shelf temperature was then raised further to 4° C. for thenext 3 hours and then finished with a 3 hour dry at room temperature.Dried samples were hydrated with 1 mL of H₂O and readily dissolved thelyophilized cake. Samples were then analyzed using Dynamic LightScattering (DLS).

Pre-frozen CPX-1 liposomes showed a mean size diameter of 110 nm (FIG.1). Liposome size immediately following lyophilization and rehydrationwere observed to be 116 nm (FIG. 2). Two samples of CPX-1 lyophilizedliposomes were stored at 5° C. for one month or six months, and theliposome size of the rehydrated compositions was observed. The meanliposome size for each was 117 nm and 123 nm, respectively (FIGS. 3 and4, respectively). FIGS. 1-3 show volume weighted distribution. FIG. 4Bshows the comparable volume weighted distribution. Results of other lesspreferred algorithms are shown in FIGS. 4A and 4C. Unless otherwisespecified, mean diameter refers to volume weighted distribution.

Experiments similar to those represented in FIGS. 1-4 were also carriedout for CPX-351 liposomes.

As noted above, CPX-351 is a liposomal formulation of a fixedcombination of the antineoplastic drugs cytarabine and daunorubicinhydrochloride. Liposomes are made using DSPC, DSPG and CHOL at a 7:2:1mol ratio and with a copper gluconate-triethanolamine buffer, pH 7.4.The crude liposomes are extruded to bring the size distribution of theliposome particles where the mean liposome diameter must be between 80nm and 110 nm with D99 not more than 200 nm (analysis by dynamic lightscattering). Cytarabine is encapsulated by a passive loading mechanism.Daunorubicin is encapsulated by an active copper-mediated mechanism toachieve a cytarabine:daunorubicin molar ratio of 5:1. Anynon-encapsulated drug substances are removed, and the bulk buffer ischanged by diafiltration. Multiple volumes of 300 mM sucrose areexchanged to finalize the CPX-351 liposomes which are then run through alyophilization optimization. Dried CPX-351 samples are reconstitutedwith 19 mL of H₂O and readily reform a liposomal dispersion. Samples arethen analyzed using Dynamic Light Scattering (DLS).

Pre-lyophilized CPX-351 liposomes showed a mean size diameter of about100 nm. Liposome size immediately following freezing and thenlyophilization/rehydration were observed to be 99 nm and 100 nm,respectively for Batch 1 (“1C001” in Table 1 below). For a second batch,1D002, CPX-351 liposome size immediately following freezing and thenlyophilization/rehydration were observed to be 104 nm and 105 nm,respectively.

These results show that DSPC/DSPG low-cholesterol liposomes co-loadedwith either irinotecan plus floxuridine or cytarabine plus daunorubicineffectively maintain their size distribution profiles upon freezing aswell as lyophilizing and for prolonged periods of storage. The resultshere also show that these gel-phase liposomes prepared withlow-cholesterol are resistant to aggregation and fusion which commonlyresults from freezing and lyophilizing particularly in the absence ofhigh levels of cholesterol.

TABLE 1 Effects of Freezing and Lyophilization on CPX-351 Liposome Size1C001 (Batch 1) 1D002 (Batch 2) Lyophilized/ Lyophilized/ CPX-351Liposomes Frozen Rehydrated Frozen Rehydrated Mean diameter (nm) 99 100104 105 D10 (nm) 68 68 74 71 D90 (nm) 135 137 137 142 D99 (nm) 178 182178 191

Example 3 Percent Drug Encapsulation Over Time Remains Unchanged inLyophilized Liposomes

Experiments were conducted in order to examine the impact of freezingand/or lyophilization and storage on the extent of drug leakage fromdual-loaded CPX-1 or CPX-351 liposomes.

The amount of encapsulated irinotecan and floxuridine in co-loaded CPX-1liposomes was measured immediately after lyophilizing (“initial”) aswell as 6 and 9 months after storage at 5° C. Stability studiesdemonstrated the percent (%) encapsulation of irinotecan to be 99%immediately after lyophilization, 97% six months after storage and 97%nine months after storage (Table 2 below). Similarly, the percentencapsulation of floxuridine was 98% immediately after lyophilizationand 95% at both six and nine months after storage at 5° C. (Table 3below).

For CPX-351 liposomes, the effects of freezing and lyophilization onpercent drug encapsulation were also studied. As seen in Table 4 below,the amount of encapsulated cytarabine in co-loaded CPX-351 liposomes wasmeasured to be 100% immediately after freezing (“Frozen”) and 98% afterlyophilization (“Lyophilized”) in two separate batches (1C001 and1D002). The percent encapsulation of daunorubicin was 99% bothimmediately after freezing and lyophilization in both batches. Drugencapsulation is also stable when CPX-351 is stored at 5° C. or 25° C.(see Tables 5 and 6).

These results clearly demonstrate that both CPX-1 and CPX-351 gel-phaseliposomes incorporating low amounts of cholesterol and a cryoprotectantin the external solution can effectively be frozen, dehydrated andreconstituted with minimal leakage of both encapsulated drugs.

TABLE 2 Percent Encapsulation of Irinotecan in Reconstituted CPX-1Liposomes Stability Intervals Test Initial 6 month 9 month Irinotecan -% 99% 97% 97% Encapsulation

TABLE 3 Percent Encapsulation of Floxuridine in Reconstituted CPX-1Liposomes Stability Intervals Test Initial 6 month 9 month Floxuridine -% 98% 95% 95% Encapsulation

TABLE 4 Percent Encapsulation of Cytarabine and daunorubicin inReconstituted CPX-351 Liposomes 1C001 1D002 Fro- Fro- CPX-351 Liposomeszen Lyophilized zen Lyophilized Cytarabine % encapsulation 100 98 100 98Daunorubicin % encapsulation 99 99 99 99

TABLE 5 CPX-351: Cytarabine Percent Encapsulation Time post-lyophilization Stored at 5° C. Stored at 25° C. Batch 1C001 Initial 9898 3 months 98 98 6 months 98 98 9 months 98 — Batch 1D001 Initial 98 983 months 98 99 6 months 99 99 9 months 98 —

TABLE 6 CPX-351: Daunorubicin Percent Encapsulation Time post-lyophilization Stored at 5° C. Stored at 25° C. Batch 1C001 Initial 9999 3 months 99 99 6 months 99 99 9 months 99 — Batch 1D001 Initial 99 993 months 99 99 6 months 99 99 9 months 99 —

1. A method to administer therapeutic and/or diagnostic agents to ananimal subject which method comprises administering to said subject areconstituted formulation of a lyophilized composition, wherein thelyophilized composition has been reconstituted in a pharmaceuticalcarrier, which lyophilized composition comprises: (a) gel-phaseliposomes that exhibit a melting phase temperature (T_(c)) of at least37° C. wherein at least two therapeutic and/or diagnostic agents arestably associated with said liposomes; and; (b) a cryoprotectantexternal to said liposomes; and wherein said liposomes containsubstantially no internal cryoprotectant, and wherein when saidlyophilized gel-phase liposomal composition is reconstituted in saidpharmaceutical carrier, the mean diameter of the liposomes is maintainedas compared to said composition prior to lyophilization and said agentsare substantially retained in the liposomes.
 2. The method of claim 1wherein said stably associated agents are at a fixed ratio and whereinwhen said composition is reconstituted said ratio of the agents changesby no more than 25% as compared to said composition prior tolyophilization.
 3. The method of claim 1 wherein the stably associatedagents are antineoplastic agents.
 4. The method of claim 3 wherein theantineoplastic agents are daunorubicin and cytarabine, or are irinotecanand floxuridine.
 5. The method of claim 4 wherein the stably associatedagents are daunorubicin:cytarabine at a fixed ratio of 1:5.
 6. Themethod of claim 1 wherein the liposome membrane comprises 50-80 mol %DSPC.
 7. The method of claim 1 wherein the mean diameter of theliposomes increases by no more than 25% after lyophilization and uponreconstitution of said liposomes as compared to said value measuredprior to lyophilization.
 8. The method of claim 1 wherein said meandiameter of the liposomes is maintained for at least 6 months uponstorage at from 5° C. to 25° C.
 9. The method of claim 1 wherein atleast 75% of each stably associated agent is retained uponreconstitution of said liposomes.
 10. The method of claim 1 wherein thesize distribution of the liposomes changes by no more than 25% afterlyophilization and upon reconstitution of said liposomes.
 11. The methodof claim 1 wherein said administering is parenteral.
 12. The method ofclaim 1 wherein the subject is human.
 13. The method of claim 3 whereinthe subject is diagnosed with cancer.
 14. The method of claim 4 whereinthe subject is diagnosed with cancer.
 15. The method of claim 5 whereinthe subject is diagnosed with cancer.